The G-type (sarin, cyclosarin, and soman) and V-type (VX and VR) organophosphonates are among the most toxic compounds known. The toxicity of these compounds is due to their ability to inactivate acetylcholine esterase, an enzyme required for proper nerve function.(1) Acetylcholine esterase breaks down the neurotransmitter acetylcholine into acetic acid and choline. Acetylcholine conducts a nerve impulse between the nerve and the muscle, stimulating the muscle. The organophosphonate binds to the hydroxyl group on a serine at a binding site on acetylcholine esterase, preventing acetylcholine from binding at that site. If acetylcholine esterase is inhibited by an organophosphonate, acetylcholine builds up at the synapses and neuromuscular junctions and the receptor is desensitized resulting in paralysis with an estimated lethal dermal exposure of about 6 milligrams of VX for an average human.
Contact with VX is about 200-fold more toxic than soman (GD) and 300-fold more toxic than sarin (GB).(2) The extreme potential for acute toxicity with VX is due, in part, to the low volatility of this compound, which allows it to persist indefinitely on common surfaces.(3) Methods currently utilized for the destruction of organophosphate nerve agents include high temperature incineration and treatment with strong base or concentrated bleach.(4,5) Medical treatment of VX toxicity is currently limited to the injection of atropine, which reduces neurological symptoms, and oximes, which can help to reactivate the inactivated acetylcholine esterase.(2) Butyrylcholine esterase, which is closely related to acetylcholine esterase, has proven effective in animal models as a stoichiometric scavenger of VX.(6) However, the large amount of enzyme required for treatment with a stoichiometric scavenger, and the limited supply of this protein, have prevented butyrylcholine esterase from being an effective antidote for medical use.(7,8)
Enzymatic hydrolysis of nerve agents provides numerous advantages over harsh physical or chemical methods of decontamination and could provide a catalytic antidote for medical use. Enzymes such as organophosphorus acid anhydrolase (OPAA), diisopropyl-fluorophospatase (DFPase), and human paraoxonase (PON1) can hydrolytically neutralize the various G-type agents, (9,10,11,12) but except for PON1, they have no activity against the V-type agents.(11)
The enzyme phosphotriesterase (PTE) is capable of hydrolyzing a wide variety of organophosphonates including both the G-type and V-type nerve agents.(13,14) A substrate for PTE, the insecticide paraoxon (FIG. 3F) has an enzymatic efficiency that approaches the limits of diffusion (kcat/Km˜108 M−1 s−1).(15)
The high toxicity and environmental persistence of VX makes the development of novel decontamination methods particularly important. PTE is capable of hydrolyzing VX. The enzymatic efficiency of PTE for VX is more than 5-orders of magnitude lower than with paraoxon. For the hydrolysis of the G-type agents by PTE, the values of kcat/Km are between 104 and 105 M−1 s−1.(13)
The G- and V-type nerve agents all contain a chiral phosphorus center where the SP-enantiomers are significantly more toxic than the corresponding RP-enantiomers.(16,17) In general, wild-type PTE preferentially hydrolyzes the RP-enantiomers of these compounds. The overall selectivity depends on the relative size of the substituents attached to the phosphorus center, with larger differences in size resulting in greater stereoselectivity.(18)
Chiral chromophoric analogues of the G-type agents have been utilized to guide the evolution of PTE for the identification of variants that prefer the more toxic SP-enantiomers of sarin, cyclosarin, and soman.(13,16,18) The catalytic activity of PTE for the more toxic SP-enantiomer of cyclosarin (GF) has been increased by more than 4-orders of magnitude.(13) The catalytic efficiencies for the hydrolysis of the more toxic SP-enantiomers by the enhanced variants of PTE for the hydrolysis of GB, GD, and GF approach 106 M−1 s−1.(13)
Unfortunately, the activity of PTE against the V-type agents is about 3-orders of magnitude lower than that with the G-type agents (kcat/Km<103 M−1 s−1). (14, 19). The net rate of VX hydrolysis by PTE is thought to be limited more by the chemistry of the leaving group than by the stereochemistry of the phosphorus center.(13,14,20) The X-ray crystal structure of PTE shows that this enzyme folds as a distorted (β/α)8-barrel and that the bulk of the active site is formed from the 8 loops that connect the core β-strands to the subsequent α-helices.(21)
The twelve residues which make up the substrate binding site of PTE can be subdivided into three pockets that accommodate the small, large and leaving-group moieties of the substrate.(21)
The residues in the active site have been shown to be largely responsible for the observed substrate specificity.(22) Loop-7 is the largest of the loops that contribute to the substrate binding site, and is known to tolerate substantial sequence variation.(18,21,23) Previous attempts to evolve PTE for the hydrolysis of VX have utilized the insecticide demeton-S with modest success.(24,25)
It would be advantageous to have enzymes that could optimize the hydrolysis of organophosphate nerve agents, including a new analogue and mutation strategies to optimize PTE for the hydrolysis of G-agents and V-agents such as VX.